Burkholderia pseudomallei (Bp), a facultative intracellular pathogen, is a tier 1 select agent due to its potential use as a bioweapon and the often fatal disease it causes, melioidosis. Melioidosis has a wide variety of symptoms that affects essentially every tissue of the body. The varieties in clinical manifestations is due to th fact that Bp is genetically diverse among species and has a wide array of virulence mechanisms used to establish disease In order to develop effective vaccines and treatment strategies, the molecular pathogenesis of Bp must be elucidated. To date, only a handful of pathogenesis mechanisms have been described, for a bacterium with a very large and diverse genome of >7 mega base pairs. Through our innovative approach of transcriptionally profiling single Bp cells at different stages of infection, we have identified four hypothetical regulators essential for complete Bp pathogenesis in vivo. To study the pathogenic processes and the genes controlled by these regulators we propose Aim 1, which will identify the regulation networks and characterize their roles in pathogenesis. This will shed light on the regulation of either known virulence pathways and/or novel virulence pathways adding to the complete understanding of the Bp intracellular lifecycle. Aim 2 proposes the mechanistic characterization of these novel transcriptional regulators by determining DNA- regulator interactions across the entire genome. This analysis will allow for identification of binding motifs for each regulator and validate their function. Taken together, the proposed aims will greatly enrich the understanding of Bp infection within the host that can lead to the development of novel vaccine and therapeutic strategies. The overall methodology used in this proposal has broad applicability to study other medically significant facultative intracellular pathogens.